USP15 promotes the apoptosis of degenerative nucleus pulposus cells by suppressing the PI3K/AKT signalling pathway

ObjectivesDegenerative disc disease is characterized by an enhanced breakdown of its existing nucleus pulposus (NP) matrix due to the dysregulation of matrix enzymes and factors. Ubiquitin‐specific protease 15 (USP15) is reported to be abnormal in certain human diseases. However, its role in NP degeneration remains unclear. Therefore, we aimed to explore the function of USP15 in degenerative NP cell specimens.MethodsWe induced gene silencing and overexpression of USP15 in degenerative NP cells using RNA interference (RNAi) and a lentiviral vector, respectively. qRT‐PCR and Western blotting were used to determine gene and protein expression levels. Cell apoptosis was analysed via flow cytometry. Protein interaction was examined by performing a co‐immunoprecipitation assay. Furthermore, the PI3K inhibitor LY294002 and agonist IGF‐1 were used to investigate the link between USP15 and AKT in NP degeneration.ResultsWe found that USP15 was up‐regulated in degenerative NP cells and that its overexpression accelerated the process of apoptosis. Moreover, USP15 expression levels negatively correlated with AKT phosphorylation in degenerative NP cells. Furthermore, targeting and silencing USP15 with miR‐338‐3p and studying its interaction with FK506‐binding protein 5 (FKBP5) revealed enhancement of FKBP5 ubiquitination, indicating that USP15 is a component of the FKBP5/AKT signalling pathway in degenerative NP cells.ConclusionsOur results show that USP15 exacerbates NP degradation by deubiquitinating and stabilizing FKBP5. This in turn results in the suppression of AKT phosphorylation in degenerative NP cells. Therefore, our study provides insights into the understanding of USP15 function as a potential molecule in the network of NP degeneration.     

LNRLMI: Linear neighbour representation for predicting lncRNA‐miRNA interactions

LncRNA and miRNA are key molecules in mechanism of competing endogenous RNAs(ceRNA), and their interactions have been discovered with important roles in gene regulation. As supplementary to the identification of lncRNA‐miRNA interactions from CLIP‐seq experiments, in silico prediction can select the most potential candidates for experimental validation. Although developing computational tool for predicting lncRNA‐miRNA interaction is of great importance for deciphering the ceRNA mechanism, little effort has been made towards this direction. In this paper, we propose an approach based on linear neighbour representation to predict lncRNA‐miRNA interactions (LNRLMI). Specifically, we first constructed a bipartite network by combining the known interaction network and similarities based on expression profiles of lncRNAs and miRNAs. Based on such a data integration, linear neighbour representation method was introduced to construct a prediction model. To evaluate the prediction performance of the proposed model, k‐fold cross validations were implemented. As a result, LNRLMI yielded the average AUCs of 0.8475 ± 0.0032, 0.8960 ± 0.0015 and 0.9069 ± 0.0014 on 2‐fold, 5‐fold and 10‐fold cross validation, respectively. A series of comparison experiments with other methods were also conducted, and the results showed that our method was feasible and effective to predict lncRNA‐miRNA interactions via a combination of different types of useful side information. It is anticipated that LNRLMI could be a useful tool for predicting non‐coding RNA regulation network that lncRNA and miRNA are involved in.     

Isoliquiritigenin ameliorates caerulein‐induced chronic pancreatitis by inhibiting the activation of PSCs and pancreatic infiltration of macrophages

Chronic pancreatitis (CP) is characterized by persistent inflammation of the pancreas that results in progressive loss of the endocrine and exocrine compartment owing to atrophy and/or replacement with fibrotic tissue. Currently, the clinical therapeutic scheme of CP is mainly symptomatic treatment including pancreatic enzyme replacement, glycaemic control and nutritional support therapy, lacking of specific therapeutic drugs for prevention and suppression of inflammation and fibrosis aggravating in CP. Here, we investigated the effect of isoliquiritigenin (ILG), a chalcone‐type dietary compound derived from licorice, on pancreatic fibrosis and inflammation in a model of caerulein‐induced murine CP, and the results indicated that ILG notably alleviated pancreatic fibrosis and infiltration of macrophages. Further in vitro studies in human pancreatic stellate cells (hPSCs) showed that ILG exerted significant inhibition on the proliferation and activation of hPSCs, which may be due to negative regulation of the ERK1/2 and JNK1/2 activities. Moreover, ILG significantly restrained the M1 polarization of macrophages (RAW 264.7) via attenuation of the NF‐κB signalling pathway, whereas the M2 polarization was hardly affected. These findings indicated that ILG might be a potential anti‐inflammatory and anti‐fibrotic therapeutic agent for CP.     

Deficiency of Macf1 in osterix expressing cells decreases bone formation by Bmp2/Smad/Runx2 pathway

Microtubule actin cross‐linking factor 1 (Macf1) is a spectraplakin family member known to regulate cytoskeletal dynamics, cell migration, neuronal growth and cell signal transduction. We previously demonstrated that knockdown of Macf1 inhibited the differentiation of MC3T3‐E1 cell line. However, whether Macf1 could regulate bone formation in vivo is unclear. To study the function and mechanism of Macf1 in bone formation and osteogenic differentiation, we established osteoblast‐specific Osterix (Osx) promoter‐driven Macf1 conditional knockout mice (Macf1^f/fOsx‐Cre). The Macf1^f/fOsx‐Cre mice displayed delayed ossification and decreased bone mass. Morphological and mechanical studies showed deteriorated trabecular microarchitecture and impaired biomechanical strength of femur in Macf1^f/fOsx‐Cre mice. In addition, the differentiation of primary osteoblasts isolated from calvaria was inhibited in Macf1^f/fOsx‐Cre mice. Deficiency of Macf1 in primary osteoblasts inhibited the expression of osteogenic marker genes (Col1, Runx2 and Alp) and the number of mineralized nodules. Furthermore, deficiency of Macf1 attenuated Bmp2/Smad/Runx2 signalling in primary osteoblasts of Macf1^f/fOsx‐Cre mice. Together, these results indicated that Macf1 plays a significant role in bone formation and osteoblast differentiation by regulating Bmp2/Smad/Runx2 pathway, suggesting that Macf1 might be a therapeutic target for bone disease.     

Protein Kinase D3 promotes the cell proliferation by activating the ERK1/c‐MYC axis in breast cancer

Breast cancer is the second leading death cause of cancer death for all women. Previous study suggested that Protein Kinase D3 (PRKD3) was involved in breast cancer progression. In addition, the protein level of PRKD3 in triple‐negative breast adenocarcinoma was higher than that in normal breast tissue. However, the oncogenic mechanisms of PRKD3 in breast cancer is not fully investigated. Multi‐omic data showed that ERK1/c‐MYC axis was identified as a major pivot in PRKD3‐mediated downstream pathways. Our study provided the evidence to support that the PRKD3/ERK1/c‐MYC pathway play an important role in breast cancer progression. We found that knocking out PRKD3 by performing CRISPR/Cas9 genome engineering technology suppressed phosphorylation of both ERK1 and c‐MYC but did not down‐regulate ERK1/2 expression or phosphorylation of ERK2. The inhibition of ERK1 and c‐MYC phosphorylation further led to the lower protein level of c‐MYC and then reduced the expression of the c‐MYC target genes in breast cancer cells. We also found that loss of PRKD3 reduced the rate of the cell proliferation in vitro and tumour growth in vivo, whereas ectopic (over)expression of PRKD3, ERK1 or c‐MYC in the PRKD3‐knockout breast cells reverse the suppression of the cell proliferation and tumour growth. Collectively, our data strongly suggested that PRKD3 likely promote the cell proliferation in the breast cancer cells by activating ERK1‐c‐MYC axis.     

Glucosamine protects against radiation‐induced lung injury via inhibition of epithelial‐mesenchymal transition

Radiotherapy is one of the most important treatments for chest tumours. Although there are plenty of strategies to prevent damage to normal lung tissues, it cannot be avoided with the emergence of radiation‐induced lung injury. The purpose of this study was to investigate the potential radioprotective effects of glucosamine, which exerted anti‐inflammatory activity in joint inflammation. In this study, we found glucosamine relieved inflammatory response and structural damages in lung tissues after radiation via HE staining. Then, we detected the level of epithelial‐mesenchymal transition marker in vitro and in vivo, which we could clearly observe that glucosamine treatment inhibited epithelial‐mesenchymal transition. Besides, we found glucosamine could inhibit apoptosis and promote proliferation of normal lung epithelial cells in vitro caused by radiation. In conclusion, our data showed that glucosamine alleviated radiation‐induced lung injury via inhibiting epithelial‐mesenchymal transition, which indicated glucosamine could be a novel potential radioprotector for radiation‐induced lung injury.     

Osthole stimulates bone formation,drives vascularization and retards adipogenesis to alleviate alcohol‐induced osteonecrosis of the femoral head

Characteristic pathological changes in osteonecrosis of the femoral head (ONFH) include reduced osteogenic differentiation of bone mesenchymal stem cells (BMSCs), impaired osseous circulation and increased intramedullary adipocytes deposition. Osthole is a bioactive derivative from coumarin with a wide range of pharmacotherapeutic effects. The aim of this study was to unveil the potential protective role of osthole in alcohol‐induced ONFH. In vitro, ethanol (50 mmol/L) remarkably decreased the proliferation and osteogenic differentiation of BMSCs and impaired the proliferation and tube formation capacity of human umbilical vein endothelial cell (HUVECs), whereas it substantially promoted the adipogenic differentiation of BMSCs. However, osthole could reverse the effects of ethanol on osteogenesis via modulating Wnt/β‐catenin pathway, stimulate vasculogenesis and counteract adipogenesis. In vivo, the protective role of osthole was confirmed in the well‐constructed rat model of ethanol‐induced ONFH, demonstrated by a cascade of radiographical and pathological investigations including micro‐CT scanning, haematoxylin‐eosin staining, TdT‐mediated dUTP nick end labelling, immunohistochemical staining and fluorochrome labelling. Taken together, for the first time, osthole was demonstrated to rescue the ethanol‐induced ONFH via promoting bone formation, driving vascularization and retarding adipogenesis.     

Melatonin against acute ischaemic stroke dependently via suppressing both inflammatory and oxidative stress downstream signallings

This study tested the hypothesis that melatonin (Mel) therapy preserved the brain architectural and functional integrity against ischaemic stroke (IS) dependently through suppressing the inflammatory/oxidative stress downstream signalling pathways. Adult male B6 (n = 6 per each B6 group) and TLR4 knockout (ie TLR4^?/?) (n = 6 per each TLR4^?/? group) mice were categorized into sham control (SC^B6), SC^TLR4?/?, IS^B6, IS^TLR4?/?, IS^B6 + Mel (i.p. daily administration) and IS^TLR4?/? + Mel (i.p. daily administration). By day 28 after IS, the protein expressions of inflammatory (HMBG1/TLR2/TLR4/MAL/MyD88/RAM TRIF/TRAF6/IKK‐α/p‐NF‐κB/nuclear‐NF‐κB/nuclear‐IRF‐3&7/IL‐1β/IL‐6/TNF‐α/IFN‐γ) and oxidative stress (NOX‐1/NOX‐2/ASK1/p‐MKK4&7/p‐JNK/p‐c‐JUN) downstream pathways as well as mitochondrial‐damaged markers (cytosolic cytochrome C/cyclophilin D/SRP1/autophagy) were highest in group IS^B6, lowest in groups SC^B6 and SC^TLR4?/?, lower in group IS^TLR4?/? + Mel than in groups IS^TLR4?/? and IS^B6 + Mel and lower in group IS^B6 + Mel than in group IS^TLR4?/? (all P < .0001). The brain infarct volume, brain infarct area and the number of inflammatory cells in brain (CD14/F4‐88) and in circulation (MPO+//Ly6C+/CD11b+//Ly6G+/CD11b+) exhibited an identical pattern, whereas the neurological function displayed an opposite pattern of inflammatory protein expression among the six groups (all P < .0001). In conclusion, TLR inflammatory and oxidative stress signallings played crucial roles for brain damage and impaired neurological function after IS that were significantly reversed by Mel therapy.